Detection of immobilized nucleic acid

ABSTRACT

The present invention provides methods for determining the presence of immobilized nucleic acid employing unsymmetrical cyanine dyes that are derivatives of thiazole orange, a staining solution and select fluorogenic compounds that are characterized as being essentially non-genotoxic. The methods comprise immobilizing nucleic acid, single or double stranded DNA, RNA or a combination thereof, on a solid or semi solid support, contacting the immobilized nucleic acid with an unsymmetrical cyanine dye compound and then illuminating the immobilized nucleic acid with an appropriate wavelength whereby the presence of the nucleic acid is determined. The cyanine dye compounds are typically present in an aqueous staining solution comprising the dye compound and a tris acetate or tris borate buffer wherein the solution facilitates the contact of the dye compound and the immobilized nucleic acid. Typically the solid or semi-solid support is selected from the group consisting of a polymeric gel, a membrane, an array, a glass bead, a glass slide, and a polymeric microparticle. Preferably, the polymeric gel is agarose or polyacrylamide. The methods employing the non-genotoxic compounds represent an improvement over commonly used methods employing ethidium bromide wherein the present methods retain the advantages of ethidium bromide, ease of use and low cost, but without the disadvantageous, known mutagen requiring special handling and waste procedures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/778,774 filed May 12, 2010, which is a divisional of U.S. patentapplication Ser. No. 10/956,868 filed Sep. 30, 2004, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/507,630filed Sep. 30, 2003, the contents of which are hereby incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to unsymmetrical cyanine monomer compoundsthat provide a detectable fluorescent signal when complexed with nucleicacid polymers. The invention has applications in the fields of molecularbiology and fluorescence based assays.

BACKGROUND OF THE INVENTION

The detection of immobilized nucleic acid, especially nucleic acidseparated on gels, is a widely used method. Numerous chromophores anddyes exist for the detection of nucleic acids however, despite itsrelatively high detection limit, ethidium bromide is still one of themost commonly used nucleic acid detection reagents due in part to itsease of use and low cost.

Ethidium bromide is easy to use as a nucleic acid gel stain because thenucleic acid can be pre- or post-stained and requires no specialequipment for visualization beyond a UV light source. Ethidium bromideis excited by UV light, less than 400 nm, and has an emission spectra ofabout 620 nm when bound to DNA. Thus, the stained gels can be excited byan ultraviolet transilluminator, which typically has a light wavelengthof about 300 nm, and the excited ethidium bromide-DNA complex gelphotographed using black and white Polaroid film. Despite theconvenience of ethidium bromide, the compound posses some significantdisadvantages; namely that the compound is a known mutagen andcarcinogen which requires special handling and waste disposalprocedures.

Ethidium bromide has been shown to inhibit replication in severalorganisms by interfering with both DNA and RNA synthesis, to bemutagenic in an Ames test and to cause frameshift mutations in bacteria(M. J. Waring J. Mol. Biol. 13 (1965) 269-282; McCann et al. PNAS 72(1975) 5135-5139; Singer et al. Mutation Research 439 (1999) 37-47).This is because ethidium bromide is believed to intercalate dsDNA andthus causes errors during replication (Fukunaga et al. Mutation Research127 (1984) 31-37).

Due to these limitations of ethidium bromide, we wanted to develop animproved method for detecting immobilized nucleic acid that retained theadvantages of ethidium bromide, ease of use and low cost, but overcamethe limitations of ethidium bromide. Thus, to satisfy this criteria themethod and subsequent dye must 1) be relatively easy to synthesize inlarge quantities (low cost), 2) be present in the staining solution at arelatively low concentration (low cost), 3) excited by UV light (ease ofuse so that the nucleic acid-dye complex can be visualized with a UVtransilluminator), 4) at least as sensitive as ethidium bromide (ease ofuse), 5) non-genotoxic (non-mutagenic and non-toxic) and 6)non-hazardous to aquatic life thus requiring no special waste disposal.

Here in we report the use of a class of unsymmetrical cyanine dyecompounds (U.S. Pat. Nos. 4,883,867 and 4,957,870) for detectingimmobilized nucleic acid polymers that is at least as sensitive asethidium bromide, requires no additional reagents or instruments thanethidium bromide and can be made in large quantities. We also report ona compound in this class of dye compounds that is non-genotoxic andtherefore requires no special handling or waste disposal procedures bythe end user. Thus, this present invention is an improvement overcurrently used nucleic acid detection reagents and solves a problem notpreviously solved.

SUMMARY OF THE INVENTION

The present invention provides methods for determining the presence ofimmobilized nucleic acid employing unsymmetrical cyanine dyes, astaining solution and select fluorogenic compounds that arecharacterized as being essentially non-genotoxic. The methods compriseimmobilizing nucleic acid, single or double stranded DNA, RNA or acombination thereof, on a solid or semi solid support, contacting theimmobilized nucleic acid with an unsymmetrical cyanine dye compound andthen illuminating the immobilized nucleic acid with an appropriatewavelength whereby the presence of the nucleic acid is determined. Thecyanine dye compounds are typically present in an aqueous stainingsolution comprising the dye compound and a tris acetate or tris boratebuffer wherein the solution facilitates the contact of the dye compoundand the immobilized nucleic acid. Typically the solid or semi-solidsupport is selected from the group consisting of a polymeric gel, amembrane, an array, a glass, and a polymeric microparticle. Preferably,the polymeric gel is agarose or polyacrylamide.

Alternatively, the invention provides methods wherein the nucleic acidis contacted with the cyanine dye compounds to pre-stain the nucleicacid and then immobilized on a solid or semi-solid support. When thismethod is used with a polymeric gel such as agarose or polyacrylamidegel the nucleic acid is pre-stained and then immobilized on the gel,typically by electrophoresis. However, the pre-stained nucleic acid mayalso be immobilized on other supports such as a glass slide or polymericbeads. In another aspect, when polymeric gels are employed the cyaninedye compounds can be mixed with unpolymerized gel and then solidified.In this method, the nucleic acid is immobilized in the gel and detectedwherein the cyanine dye binds the nucleic acid producing a fluorescentdetectable signal. The cyanine dye compounds of the present methods arefluorogenic, they have a low intrinsic fluorescence when not associatedwith nucleic acid, but when bound to or associated with nucleic acidbecome fluorescent. This is an improvement over ethidium bromide whereinthe compound has significant intrinsic fluorescence and displays a20-25-fold increase in fluorescence upon intercalating into doublestranded regions of nucleic acid (J. B. LePecq Anal. Biochem. 17 (1966)100-107).

The cyanine dye compounds of the present invention include any compounddisclosed in U.S. Pat. Nos. 4,883,867 and 4,957,870, supra. Thesecyanine dye compounds have the following formula

wherein X is O, S or C(CH₃)₂, R¹ is a fused benzene, C₁-C₆ alkoxy, or aC₁-C₆ alkyl, R² and R³ are independently a C₁-C₆ alkyl and R⁴ is a C₁-C₆alkyl or a C₁-C₆ alkoxy, wherein t is independently 0, 1, 2, 3, or 4 ands is independently 0, 1, 2, 3, or 4. n is 0, 1, 2 or 3, with the provisothat the dye is not thiazole orange when used to detect DNA in a gel.

These cyanine dye compounds have previously been disclosed for use indetecting reticulocytes in a blood sample but herein we report a noveluse for these compounds as fluorogenic dyes for immobilized nucleic acidpolymers. In a preferred embodiment the cyanine dye compounds areemployed as gel stains for nucleic acid polymers separated byelectrophoresis, preferably DNA.

We have unexpectedly found that certain select unsymmetrical cyanine dyecompounds can be characterized as being essentially non-genotoxic. Themost widely used DNA gel stain is ethidium bromide, however thiscompound is a known mutagen and thus requires special handling and wastedisposal. We herein report on an unsymmetrical cyanine dye compound thatis at least as sensitive as ethidium bromide and based on an Ames test,in vitro transformation test, forward mutation screen and a screen forchromosomal aberrations is essentially non-mutagenic and non-toxic(Examples 2-5).

Therefore, identification of an essentially non-genotoxic dye compoundthat is at least as sensitive as ethidium bromide overcomes thelimitations of ethidium bromide by solving the problem of specialhandling and waste disposal not previously solved (See, Example 7). Inaddition, the dye compounds are excited by UV light, are easy to use andsynthesize in large quantities. The identification of non-genotoxiccompounds provides a DNA gel stain that does not poses a mutagenic ortoxic hazard to the end user. This was an unexpected finding becausecompounds that bind or associate with nucleic acid are considered aspotential mutagens by possibly interfering with replication.

For comparison purposes the compounds thiazole orange, ethidium bromideand a compound having the formula

were tested for their ability to induce genetic mutations and toxicitylevels in cells. The tests demonstrated that Compound 1, a thiazoleorange derivative, is characterized as being essentially non-genotoxicwhile thiazole orange can not be characterized as such based on thetests performed. Therefore, this compound is preferred for the detectionof immobilized nucleic acid wherein the compound posses no genotoxic(mutagenic or toxic) hazard to the end user.

Thus, the present invention provides improved methods for the detectionof immobilized nucleic acid employing thiazole orange derivative cyaninedye compounds of the present invention and an aqueous staining solution.A particularly preferred improvement is the use of Compound 1 for thedetection of immobilized nucleic acid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the detection of DNA, pre- (FIGS. 1A and D) andpost-stained (FIGS. 1C and B), in an agarose gel using thiazole orange(FIGS. 1A and B) and Compound 1 (FIGS. 1C and D). See, Example 1

FIG. 2: Shows the comparison between ethidium bromide and Compound 1 inthe Ames test. See, Example 2.

FIG. 3: shows a comparison between DNA stained with ethidium bromide(FIG. 1A) and Compound 1 (FIG. 1B-D) wherein FIGS. 1A and B are poststained for 30 minutes and FIG. 1C is post stained for 60 minutes andFIG. 1D for 90 minutes. See, Example 6.

FIG. 4: Shows the detection of DNA on an E-gel visualized with a UVtransilluminator and a Dark Reader (Clare Chemical Research). Differentquantities of Low DNA Mass Ladder (1 μl, 0.5 μl, 0.25 μl, 0.13 μl), wereloaded on an E-Gel (2%), where the ethidium bromide has been replaced bya 4× concentration of Compound 1. The gels were run for 30 minutes, thenvisualized with a transilluminator. See, Example 9.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a cyanine dye compound”includes a plurality of compounds and reference to “nucleic acid”includes a plurality of nucleic acids and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

Although typically not shown for the sake of clarity, any overallpositive or negative charges possessed by any of the compounds of theinvention are balanced by a necessary counterion or counterions. Wherethe compound of the invention is positively charged, the counterion istypically selected from, but not limited to, chloride, bromide, iodide,sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylborate, nitrate, hexafluorophosphate, andanions of aromatic or aliphatic carboxylic acids. Where the compound ofthe invention is negatively charged, the counterion is typicallyselected from, but not limited to, alkali metal ions, alkaline earthmetal ions, transition metal ions, ammonium or substituted ammoniumions. Preferably, any necessary counterion is biologically compatible,is not toxic as used, and does not have a substantially deleteriouseffect on biomolecules. Counterions are readily changed by methods wellknown in the art, such as ion-exchange chromatography, or selectiveprecipitation.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty-five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen,phosphorous and sulfur atoms are optionally oxidized, and the nitrogenheteroatom is optionally be quaternized, and the sulfur atoms areoptionally trivalent with alkyl or heteroalkyl substituents. Theheteroatom(s) O, N, P, S and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 4 rings), which are fused together or linked covalently.Specific examples of aryl substituents include, but are not limited to,substituted or unsubstituted derivatives of phenyl, biphenyl, o-, m-, orp-terphenyl, 1-naphthyl, 2-naphthyl, 1-, 2-, or 9-anthryl, 1-, 2-, 3-,4-, or 9-phenanthrenyl and 1-, 2- or 4-pyrenyl. Preferred arylsubstituents are phenyl, substituted phenyl, naphthyl or substitutednaphthyl.

The term “heteroaryl” as used herein refers to an aryl group as definedabove in which one or more carbon atoms have been replaced by anon-carbon atom, especially nitrogen, oxygen, or sulfur. For example,but not as a limitation, such groups include furyl, tetrahydrofuryl,pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl,isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl,pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl,pyridyl, pyridaziyl, triazinyl, piperidinyl, morpholinyl,thiomorpholinyl, pyrazinyl, piperainyl, pyrimidinyl, naphthyridinyl,benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl,quinolizinyl, qunolinyl, isoquinolinyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl,acridinyl, phenazinyl, phenothizinyl, phenoxazinyl, purinyl,benzimidazolyl and benzthiazolyl and their aromatic ring-fused analogs.Many fluorophores are comprised of heteroaryl groups and include,without limitations, xanthenes, oxazines, benzazolium derivatives(including cyanines and carbocyanines), borapolyazaindacenes,benzofurans, indoles and quinazolones.

Where a ring substituent is a heteroaryl substituent, it is defined as a5- or 6-membered heteroaromatic ring that is optionally fused to anadditional six-membered aromatic ring(s), or is fused to one 5- or6-membered heteroaromatic ring. The heteroaromatic rings contain atleast 1 and as many as 3 heteroatoms that are selected from the groupconsisting of O, N or S in any combination. The heteroaryl substituentis bound by a single bond, and is optionally substituted as definedbelow.

Specific examples of heteroaryl moieties include, but are not limitedto, substituted or unsubstituted derivatives of 2- or 3-furanyl; 2- or3-thienyl; N-, 2- or 3-pyrrolyl; 2- or 3-benzofuranyl; 2- or3-benzothienyl; N-, 2- or 3-indolyl; 2-, 3- or 4-pyridyl; 2-, 3- or4-quinolyl; 1-, 3-, or 4-isoquinolyl; 2-, 4-, or 5-(1,3-oxazolyl);2-benzoxazolyl; 2-, 4-, or 5-(1,3-thiazolyl); 2-benzothiazolyl; 3-, 4-,or 5-isoxazolyl; N-, 2-, or 4-imidazolyl; N-, or 2-benzimidazolyl; 1- or2-naphthofuranyl; 1- or 2-naphthothienyl; N-, 2- or 3-benzindolyl; 2-,3-, or 4-benzoquinolyl; 1-, 2-, 3-, or 4-acridinyl. Preferred heteroarylsubstituents include substituted or unsubstituted 4-pyridyl, 2-thienyl,2-pyrrolyl, 2-indolyl, 2-oxazolyl, 2-benzothiazolyl or 2-benzoxazolyl.

The above heterocyclic groups may further include one or moresubstituents at one or more carbon and/or non-carbon atoms of theheteroaryl group, e.g., alkyl; aryl; heterocycle; halogen; nitro; cyano;hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl- or arylthio;amino, alkyl-, aryl-, dialkyl-, diaryl-, or arylalkylamino;aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl substituents may be combined to form fused heterocycle-alkylring systems. Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The term “heterocycloalkyl” as used herein refers to a heterocycle groupthat is joined to a parent structure by one or more alkyl groups asdescribed above, e.g., 2-piperidylmethyl, and the like. The term“heterocycloalkyl” refers to a heteroaryl group that is joined to aparent structure by one or more alkyl groups as described above, e.g.,2-thienylmethyl, and the like.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)=NR″″,—NR—C(NR′R″)=NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)=NR″″,—NR—C(NR′R″)=NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

The aryl and heteroaryl substituents described herein are unsubstitutedor optionally and independently substituted by H, halogen, cyano,sulfonic acid, carboxylic acid, nitro, alkyl, perfluoroalkyl, alkoxy,alkylthio, amino, monoalkylamino, dialkylamino or alkylamido.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(ORR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

The term “amino” or “amine group” refers to the group —NR′R″ (or NRR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NRR′R″ and itsbiologically compatible anionic counterions.

The term “affinity” as used herein refers to the strength of the bindinginteraction of two molecules, such as an antibody and a ligand orantigen or a positively charged moiety and a negatively charged moiety.For bivalent molecules such as antibodies, affinity is typically definedas the binding strength of one binding domain for the antigen, e.g. oneFab fragment for the antigen. The binding strength of both bindingdomains together for the antigen is referred to as “avidity”. As usedherein “High affinity” refers to a ligand that binds to an antibodyhaving an affinity constant (K_(a)) greater than 10⁴ M⁻¹, typically10⁵-10¹¹ M⁻¹; as determined by inhibition ELISA or an equivalentaffinity determined by comparable techniques such as, for example,Scatchard plots or using K_(d)/dissociation constant, which is thereciprocal of the K_(a), etc.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “complex” as used herein refers to the association of two ormore molecules, usually by non-covalent bonding.

The term “cyanine monomer” or “cyanine dye” as used herein refers to afluorogenic compound that comprises 1) a substituted benzazolium moiety,2) a polymethine bridge and 3) a substituted or unsubstituted pyridiniumor quinolinium moiety. These monomer or dye moieties are capable offorming a non-covalent complex with nucleic acid and demonstrating anincreased fluorescent signal after formation of the nucleic acid-dyecomplex.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters.

The term “essentially non-genotoxic” as used herein refers to asubstance that causes an insignificant amount of toxicity or mutationsto a prokaryotic and/or eukaryotic cell when in contact with the cells.The non-genotoxic effect of a substance is determined by tests andscreening assays well known in the art including, but not limited to, anAmes test, chromosomal aberration test, forward mutation screen and atest that determines LC₅₀ values.

The term “genotoxic” as used herein refers to a substance that causestoxicity and/or mutations to the prokaryotic and or eukaryotic cellsresulting in abnormal cell growth including death and uncontrolledgrowth of the cell or organism.

The term “kit” as used refers to a packaged set of related components,typically one or more compounds or compositions.

The term “mutagenic” as used herein refers to a substance that causesmutations to the nucleic acid of a cell or organism including pointmutations, frameshift mutations and deletion mutations.

The term “nucleic acid polymer” as used herein refers to natural orsynthetic polymers of DNA or RNA that are single, double, triple orquadruple stranded. Polymers are two or more bases in length. The term“nucleic acid” is herein used interchangeably with “nucleic acidpolymer”.

The term “sample” as used herein refers to any material that may containa target nucleic acid. Typically, the sample is immobilized on a solidor semi solid surface such as a polyacrylamide gel, membrane blot or ona microarray that contains nucleic acid polymers, nucleotides,oligonucleotides, but may be in an aqueous solution or a viable cellculture. However, the sample may be a live cell, a biological fluid thatcomprises endogenous host cell proteins, peptides and buffer solutions.

Compound and Compositions

The present invention provides improved methods for determining thepresence of immobilized nucleic acid, an aqueous staining solution andnucleic acid complexing compounds. In one aspect of the invention, theimprovement consists of the use of cyanine dye compounds that arecharacterized as being essentially non-genotoxic. These compounds are animprovement over currently used nucleic acid detection agents that aregenerally considered to be toxic and/or mutagenic and poses a healthrisk to the end user and environment wherein precautionary measures needto be followed to ensure there is no direct contact between the nucleicacid detection reagent, such as ethidium bromide, and the user. Thus,the discovery of a non-genotoxic nucleic acid detection agent is animportant improvement that is safe to handle for the end user and can bedisposed of as non-hazardous waste, i.e. safe to the environment(Example 7). Herein we report an improved method for the detection ofnucleic acid that does not require special handling or waste disposalbut retains all of the advantages of commonly used ethidium bromide.

A number of different classes of compounds were tested that are known orthought to associate with immobilized DNA. Typically, the nucleic acidcomplexing compound are unsymmetrical cyanine dyes including, but arenot limited to, dyes sold under the trade name SYBR® dyes (MolecularProbes, Inc.), thiazole orange, their derivatives and any monomercompound disclosed in U.S. Pat. Nos. 4,957,870; 4,883,867; 5,436,134;5,658,751, 5,534,416 and 5,863,753. These compounds were simultaneouslyscreened in an Ames test for their ability to induce mutations inSalmonella typhimurium wherein the goal was to develop an improvedmethod for the detection of immobilized nucleic acid such that the dyecompound employed was at least, or more, sensitive than ethidium bromidebut with reduced genotoxic effects compared to ethidium bromide. Theresults of these early screens indicated that two compounds, thiazoleorange and Compound 1, were either mildly mutagenic or non-mutagenic andthat both were able to detect nucleic acid that had been immobilized ina gel by electrophoresis when excited with UV light, about 300 nm, SeeExample 1. For comparison purposes ethidium bromide was tested withThiazole orange and Compound 1 along with the appropriate controls, SeeExample 2-5.

Thus, in one aspect of the invention, compounds disclosed in U.S. Pat.Nos. 4,883,867 and 4,957,870 (supra) are preferred for use indetermining the presence of immobilized nucleic acid. These cyanine dyecompounds have the following formula

wherein X is O, S or C(CH₃)₂, R¹ is a fused benzene, C₁-C₆ alkoxy, or aC₁-C₆ alkyl, R² and R³ are independently a C₁-C₆ alkyl and R⁴ is a C₁-C₆alkyl or a C₁-C₆ alkoxy, wherein t is independently 0, 1, 2, 3, or 4 ands is independently 0, 1, 2, 3 or 4. n is 0, 1, 2 or 3.

In a preferred embodiment the dye compound is either thiazole orange

or Compound 1 having the formula:

wherein R¹ is hydrogen, R² is methyl, n is 0, R⁴ is hydrogen and R³ iseither methyl (thiazole orange) or propyl (Compound 1). However, the useof thiazole orange for detection of DNA in a gel is not an aspect of thepresent invention (Rye et al. Nucleic Acids Res. 19(2) (1991) 327-33).Therefore, Compound 1 is preferred for the detection of nucleic acidthat has been immobilized on a polymeric gel.

These dyes have a low intrinsic fluorescence but upon binding to nucleicacid demonstrate significant increase in fluorescence. These dyecompounds have a maxima excitation between 480 and 520 nm, however thesecompounds may be excited by UV light, which is typically understood tobe below 400 nm. Thus, the cyanine dye compounds can be excited using aUV transilluminator, as is typically used for ethidium bromide stainedgels containing separated nucleic acid. The excitation of these dyes istypically in the range of about 530 to 600 nm. Ethidium bromide can beexcited by UV light but has an optima absorption of 540 nm, whenassociated with DNA, and an emission of 620 nm. Thus, the presentcyanine dye compounds, including Compound 1, fit the criteria of beingexcitable by UV light and possessing similar excitation emissioncompared to ethidium bromide.

Thiazole orange and Compound 1 were tested in an Ames test and comparedto previously tested ethidium bromide (Singer et al. (1999) supra)(Example 2). All three compounds, ethidium bromide, Thiazole orange andCompound 1, were tested in an in vitro transformation test (Example 3),forward mutation screen (Example 4) and a screen for chromosomalaberrations (Example 5). This panel of tests results in theidentification of compounds that are either genotoxic or non-genotoxicwherein genotoxic is defined to include both cell cytoxicity effects andgenetic mutations. It is appreciated by one skilled in the art thatthese tests can be used to screen other compounds for their genotoxiceffects that are to be used to detect immobilized nucleic acid polymers.Unexpectedly, based on these tests, thiazole orange is consideredgenotoxic but the thiazole analog Compound 1 is characterized as beingessentially non-genotoxic. In addition, Compound 1 was tested todetermine if the compound is hazardous or toxic to aquatic life whereinCompound 1 has an LC₅₀ value >500 mg/L and is characterized as beingnon-hazardous to aquatic life (Example 7).

Based on the tests performed, Compound 1 does not cause mutations inmouse lymphoma cells at the thymidine kinase (TK) locus, nor does itinduce chromosomal aberrations in cultured human peripheral bloodlymphocytes, with or without S9 metabolic activation. In addition,Compound 1 did not transform Syrian hamster embryo (SHE) cell cultures.This latter test has a high concordance (>80%) with rodentcarcinogenesis, so a negative test strongly indicates that Compound 1 isnoncarcinogenic. Thus, Compound 1 is not a dangerous laboratory reagentby three independent assessments of potential genotoxicity to mammaliancells. In contrast, ethidium bromide tests positive in the SHE assay,indicating that this stain will be found carcinogenic to rodents.Two-year bioassay studies for ethidium bromide have not yet beenreported.

TABLE 1 Ethidium Thiazole Test* bromide Compound 1 orange TransformationTest [1] Syrian positive negative positive hamster embryo (SHE) cellsChromosomal Aberrations Test [2] negative negative negative Culturedhuman peripheral blood lymphocytes Forward Mutation Test [3] negativenegative negative L5178YTK^(+/−) mouse lymphoma cells [1] Yamasaki(1996) Fundamental and Molecular Mechanisms of Mutagenesis Special Issue356 1-128; [2] Evans (1976) Cytological Methods for Detecting ChemicalMutagens in Chemical Mutagens, Principles and Methods for theirDetection, Hollaender (ed). Vol. 4: 1-29; [3] Amacher et al (1980)Mutation Research 72: 447-474; Clive et al (1979) Mutation Research 59:61-108.

Compound 1 causes fewer mutations in the Ames test, compared to ethidiumbromide and thiazole orange, as measured in several different strains ofSalmonella typhimurium, See FIG. 1 and Table 2. Weakly positive results(Compound 1) in this test occurred in three out of seven strains andonly after activation by a mammalian S9 fraction obtained from ratliver, as shown in FIG. 1.

Methods of Use

The staining solution can be prepared in a variety of ways, which aredependent on the method and the medium in which the sample is present,as described below. Specifically the staining solution comprises apresent unsymmetrical cyanine dye and buffering components that arecompatible with nucleic acid, optionally the staining solution comprisesan organic solvent or a mixture of organic solvents and additional ionicor nonionic components. Any of the components of the staining solutioncan be added together or separately and in no particular order and, aswill become evident, the cyanine dye compound may be immobilized on asolid or semi-solid matrix, wherein the buffering components are addedto the matrix to form the staining solution of the present invention.Therefore, the cyanine compounds do not need to free in the stainingsolution to form the solution but may be immobilized on a solid orsemi-solid matrix surface. Alternatively the cyanine compounds areimmobilized on or in a solid or semi-solid matrix wherein the dyecompound is transferred to the immobilized nucleic acid in the absenceof a buffer. In another aspect the cyanine dye compound is immobilizedin a polymeric gel that is a buffer-less system such as E-gels(Inivtrogen Corp).

The staining solution is typically prepared by dissolving a presentunsymmetrical cyanine dye compound in an aqueous solvent such as water,a buffer solution, such as phosphate buffered saline, or an organicsolvent such as dimethylsulfoxide (DMSO), dimethylformamide (DMF),methanol, ethanol or acetonitrile. Typically, the present cyanine dyecompounds are first dissolved in an organic solvent such as DMSO as astock solution. Typically the stock solution is about 100-fold to about10.000-fold concentrated compared to the working concentration.

In one aspect, the stock solution is then diluted to an effectiveworking concentration in an aqueous solution optionally comprisingappropriate buffering components to form a buffer solution comprising adye compound of the present invention and a trace amount of the organicsolvent. The buffer solution is typically phosphate buffered saline(PBS), tris acetate (TAE) or tris borate (TBE). Preferably the stainingsolution comprises a cyanine dye of the present invention, TAE or TBEand a trace amount of DMSO. An effective working concentration of thepresent compounds is the amount sufficient to give a detectablefluorescent response when complexed with nucleic acid polymers.Typically, the effective amount is about 100 nm to 100 μM. Preferred isabout 600 nm to 10 μM and most preferred is about 1 μM. It is generallyunderstood that the specific amount of the dye compound present in astaining solution is determined by the physical nature of the sample andthe nature of the analysis being performed.

An aqueous staining solution of the present invention for determiningthe presence of immobilized nucleic acid on a solid or semi-solidsupport wherein the nucleic acid is essentially free of intact cells orcellular organelles, comprises an unsymmetrical cyanine dye compound ofthe present invention, a tris borate or tris acetate buffer and a traceamount of organic solvent that was used to solubilize the dye compound.The staining solution typically has a pH of about 6 to about 8 and thesolid or semi-solid support is selected from the group consisting of apolymeric gel, a membrane, an array, a glass, and a polymericmicroparticle. In one aspect of the invention, the solution optionallyfurther comprises unpolymerized agarose or polyacrylamide such that thedye compound forms part of the gel and the nucleic acid sample comes incontact with the dye compound when immobilized on the gel.

Thus, the dye stock solution is diluted and mixed with agarose and/oragarose and buffer, wherein the nucleic acid is immobilized in theagarose that contains a present compound. The agarose may be in the formof a tablet, pre-cast gel or solidified agarose that is ready to beheated and poured into a slab gel. One possible form of this would be amixture of agarose/TBE/and a present compound at the concentrations thatwould be used for electrophoretic separation of nucleic acids. Heatingthe mixture until molten, mixing, and allowing to cool to roomtemperature. At anytime in the future the solid mixture may be reheatedand poured for use without the need for measuring of components ormixing prior to use. Another iteration of this concept would be to blendthe solid form of the dye with solid powdered agarose (which can eitherbe stored as a powder or compressed into tablets) and stored. Whenneeded the powder may be weighed and added to buffer for use without theneed to separately measure and add the dye.

In yet another aspect, the present compounds are impregnated in apolymeric membrane, such as InstStain papers (Edvotek), wherein themembrane is contacted with the immobilized nucleic acid resulting in atransfer of the dye from the membrane to the nucleic acid.

In one aspect of the invention, a method for determining the presence orabsence of nucleic acid immobilized on a solid or semi solid supportcomprises

-   -   a) combining an unsymmetrical cyanine dye compound of the        present invention with a sample to prepare a labeling mixture,        wherein the sample is immobilized on a solid or semi-solid        support;    -   b) incubating the labeling mixture for a sufficient amount of        time for the dye to associate with the nucleic acid to prepare        an incubated sample;    -   c) illuminating the incubated sample with an appropriate        wavelength to prepare an illuminated sample; and    -   d) observing the illuminated sample whereby the presence or        absence of the nucleic acid is determined.

In one aspect of the invention, the nucleic acids in the sample mixtureare separated from each other or from other ingredients in the sample bymobility (e.g. electrophoretic gel or capillary) or by size (e.g.centrifugation, pelleting or density gradient), or by binding affinity(e.g. to a filter membrane) in the course of the method. The sample iscombined with the staining solution by any means that facilitatescontact between the dye and the analyte. Thus, the present compounds maybe staining solution, dried on a polymeric membrane or pre-mixed withthe solid or semi-solid support that the nucleic acid is immobilized on.Typically the contact occurs through simple mixing, as in the case wherethe sample is a solution. A staining solution containing the dye may beadded to the analyte solution directly or may contact the analytesolution in a liquid separation medium such as an electrophoreticliquid, sieving matrix or running buffer, or in a sedimentation (e.g.sucrose) or buoyant density gradient (e.g. containing CsCl), or on aninert matrix, such as a blot or gel, a testing strip, or any other solidor semi-solid support. Suitable supports also include, but are notlimited to, polymeric microparticles (including paramagneticmicroparticles), polyacrylamide and agarose gels, nitrocellulosefilters, computer chips (such as silicon chips), natural and syntheticmembranes, and glass (including optical filters), and other silica-basedand plastic support. The dye is optionally combined with the analytesolution prior to undergoing gel or capillary electrophoresis, gradientcentrifugation, or other separation step, during separation, or afterthe nucleic acids undergo separation. Alternatively, the dye is combinedwith an inert matrix or solution in a capillary prior to addition of theanalyte solution, as in pre-cast gels, capillary electrophoresis orpreformed density or sedimentation gradients.

The sample is incubated in the presence of the dye compounds for a timesufficient to form the fluorescent nucleic acid-dye compound complex.Detectable fluorescence in a solution of nucleic acids is essentiallyinstantaneous. In general, visibly detectable fluorescence can beobtained in a wide variety of solid or semi-solid matrix withembodiments of the present invention within about 10-90 minutes aftercombination with the sample, commonly within about 20-60 minutes, mostpreferably about 30 minutes (See, Example 6). In this instance, anucleic acid sample is immobilized on a polymeric gel, typically byelectrophoresis, and then the gel is immersed in the staining solutionwherein a detectable signal represents the presence of nucleic acid. Itis readily apparent to one skilled in the art that the time necessaryfor sufficient formation of the fluorescent nucleic acid complex isdependent upon the physical and chemical nature of the individual sampleand the sample medium.

In an alternative embodiment, the immobilized nucleic acid is overlaidwith a membrane that contains a present cyanine dye compound. Thecompound transfers to the nucleic acid in a few minutes, typically lessthan about 10 minutes, to provided a labeled sample mixture.

To facilitate the detection of the nucleic acid-dye compound complex,the excitation or emission properties of the fluorescent complex areutilized. For example, the sample is excited or illuminated by a lightsource capable of producing light at or near the wavelength of maximumabsorption of the fluorescent complex, such as an ultraviolet or visiblelamp, an arc lamp, a laser, or even sunlight. Preferably the fluorescentcomplex is excited at a wavelength equal to or greater than about 280nm, more preferably equal to or greater than about 300 nm. The resultingemission is detected by means that include visible inspection,photographic film, or the use of current instrumentation such asfluorometers, quantum counters, plate readers, epifluorescencemicroscopes and flow cytometers or by means for amplifying the signalsuch as a photomultiplier. In one aspect a UV transilluminator is usedto illuminate the nucleic acid-dye compound complex. In another aspect avisible light transilluminator, such as a Dark Reader (Clare ChemicalResearch, Inc., CO) is used to illuminate the nucleic acid-dye compoundcomplex.

In one aspect of the invention a method for determining the presence ofnucleic acid polymer immobilized on a gel comprises the following steps:

-   -   a) immobilizing the nucleic acid polymers on a polymeric gel;    -   b) contacting the gel with a staining solution, wherein the        staining solution comprises;        -   i) an unsymmetrical cyanine dye compound having formula

-   -   -   -   wherein X is O, S or C(CH₃)₂;            -   R¹ is a fused benzene, methoxy, a C₁-C₆ alkyl;            -   R² and R³ are independently a C₁-C₆ alkyl;            -   R⁴ is a C₁-C₆ alkyl or a methoxy wherein t is                independently            -   0, 1, 2, 3, or 4, and            -   n is 0, 1, 2 or 3; and

        -   ii) a tris borate or tris acetate buffer,

    -   c) incubating the gel of step b) and the staining solution for        sufficient time to allow the cyanine dye compound to associate        with the nucleic acid polymer; and,

    -   d) illuminating the immobilized nucleic acid-cyanine dye complex        with an appropriate wavelength whereby the presence of the        nucleic acid is determined, with the provision that the cyanine        dye compound is not thiazole orange.

Typically the gel is about a 3-0.5% agarose gel. Preferably the agarosegel is about a 1% gel. However, one of skill in the art will appreciatethat the percentage of gel is somewhat dependent on the size of thenucleic acid polymers to be separated and immobilized.

The nucleic acid polymers are typically immobilized by electrophoresiswherein a current is applied to the agarose gel and the charged nucleicacid polymer migrate through the gel as a function of size. However, thenucleic acid polymers may be spotted onto the polymeric gel, typicallyagarose or polyacrylamide. In one aspect agarose E-gels (Invitrogen, CA)are used to separate and immobilize a sample containing nucleic acid.

In a preferred embodiment the cyanine dye compound is represented byCompound 1. In this instance, Compound 1 is characterized as beingessentially non-gentoxic. Therefore, a preferred embodiment of thepresent invention is the improved method of determining the presence ofnucleic acid using a compound that is essentially non-genotoxic. Thismethod provides an improvement over currently used dye compounds todetect nucleic acid wherein compound 1 has not previously been disclosedto be non-genotoxic or disclosed for the use of detecting immobilizednucleic acid.

As described above, the gel is contacted with a present stainingsolution, typically by immersing the gel in the staining solution. Thegel is typically immersed in the staining solution for about 10-90minutes, preferably about 20-60 minutes, most preferred about 30minutes. However, the running buffer, buffer used to conduct the currentthrough the gel may be replaced by the staining solution of the presentinvention. In this instance, the nucleic acid is forming a complex withthe cyanine dye while it is migrating through the gel. In this way, thestep of incubating occurs simultaneously with the step of immobilizingand the step of contacting.

The stained agarose gel is typically illuminated with a UVtransilluminator or a visible light transilluminator. However, anyappropriate instrument that allows for visualization of the nucleicacid-dye complex, excites the fluorophore and records the excitedwavelength generated by the fluorophore, may be used for the detectionof the nucleic acid-dye complex.

In another aspect of the invention, the staining solution, comprising anunsymmetrical cyanine dye compound of the present invention, is combinedwith the unpolymerized gel such that the cyanine dye compound forms partof the solidified gel. In this instance, dry agarose is combined withbuffer such as TBE and heated to dissolve the dry agarose. Prior tosolidifying a stock solution of the cyanine dye compound is added to theliquefied agarose in buffer. Therefore, the steps of immobilizing,contacting and incubating occur simultaneously in this aspect of theinvention.

Sample Preparation

The end user will determine the choice of the sample and the way inwhich the sample is prepared but the sample is typically prepared usingmethods well known in the art for isolating nucleic acid for in vitrosolution based assay detection or well know methods for detection ofnucleic acids that have been immobilized on a solid or semi-solidmatrix. The sample includes, without limitation, any biological derivedmaterial that is thought to contain a nucleic acid polymer.Alternatively, samples also include material that nucleic acid polymershave been added to such as a PCR reaction mixture, a polymer gel such asagarose or polyacrylamide gels or a microfluidic assay system. Inanother aspect of the invention, the sample can also include a buffersolution that contains nucleic acid polymers to determine the presentdye compounds that are ideal under different assay conditions or todetermine the present dye compounds that are essentially non-genotoxic.

The sample can be a biological fluid such as whole blood, plasma, serum,nasal secretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. Biological fluids also include tissueand cell culture medium wherein an analyte of interest has been secretedinto the medium. Alternatively, the sample may be whole organs, tissueor cells from the animal. Examples of sources of such samples includemuscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver,kidney, spleen, thymus, pancreas, solid tumors, macrophages, mammaryglands, mesothelium, and the like. Cells include without limitationprokaryotic cells such as bacteria, yeast, fungi, mycobacteria andmycoplasma, and eukaryotic cells such as nucleated plant and animalcells that include primary cultures and immortalized cell lines.Typically prokaryotic cells include E. coli and S. aureus. Eukaryoticcells include without limitation ovary cells, epithelial cells,circulating immune cells, 13 cells, hepatocytes, and neurons.

The nucleic acid may be either natural (biological in origin) orsynthetic (prepared artificially). The nucleic acid may be present asnucleic acid fragments, oligonucleotides, or nucleic acid polymers. Thenucleic acid may be present in a condensed phase, such as a chromosome.The presence of the nucleic acid in the sample may be due to asuccessful or unsuccessful experimental methodology, undesirablecontamination, or a disease state. Nucleic acid may be present in all,or only part, of a sample, and the presence of nucleic acids may be usedto distinguish between individual samples, or to differentiate a portionor region within a single sample.

The nucleic acid may be enclosed in a biological structure, for examplecontained within a viral particle, an organelle, or within a cell. Thenucleic acids enclosed in biological structures may be obtained from awide variety of environments, including cultured cells, organisms ortissues, unfiltered or separated biological fluids such as urine,cerebrospinal fluid, blood, lymph fluids, tissue homogenate, mucous,saliva, stool, or physiological secretions or environmental samples suchas soil, water and air. The nucleic acid may be endogenous or introducedas foreign material, such as by infection or by transfection.

Alternatively, the nucleic acid is not enclosed within a biologicalstructure, but is present as a sample solution. The sample solution canvary from one of purified nucleic acids to crude mixtures such as cellextracts, biological fluids and environmental samples. In some cases itis desirable to separate the nucleic acids from a mixture ofbiomolecules or fluids in the solution prior to combination with thepresent cyanine dye compounds. Numerous, well known, techniques existfor separation and purification of nucleic acids from generally crudemixtures with other proteins or other biological molecules. Theseinclude such means as electrophoretic techniques and chromatographictechniques using a variety of supports.

Illumination

The sample containing a nucleic acid-dye compound complex is illuminatedwith a wavelength of light selected to give a detectable opticalresponse, and observed with a means for detecting the optical response.Equipment that is useful for illuminating the present compounds andcompositions of the invention includes, but is not limited to, a UVtansilluminator, hand-held ultraviolet lamps, mercury arc lamps, xenonlamps, lasers, laser diodes and a Dark Reader or any transilluminaterdisclosed in U.S. Pat. Nos. 6,512,236 and 6,198,107. These illuminationsources are optically integrated into laser scanners, fuorescencesmicroplate readers or standard or microfluorometers.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD camera, video camera,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes.

The wavelengths of the excitation and emission bands of the nucleic aciddye compounds vary with dye compound composition to encompass a widerange of illumination and detection bands. This allows the selection ofindividual dye compounds for use with a specific excitation source ordetection filter.

Kits

Suitable kits for forming a nucleic acid-dye compound complex anddetecting the nucleic acid also form part of the invention. Such kitscan be prepared from readily available materials and reagents and cancome in a variety of embodiments. The contents of the kit will depend onthe design of the assay protocol or reagent for detection ormeasurement. All kits will contain instructions, appropriate reagents,and present nucleic acid dye compounds. Typically, instructions includea tangible expression describing the reagent concentration or at leastone assay method parameter such as the relative amounts of reagent andsample to be added together, maintenance time periods for reagent/sampleadmixtures, temperature, buffer conditions and the like to allow theuser to carry out any one of the methods or preparations describedabove.

In one aspect of the invention, a kit contains a solution that comprisesan organic solvent and an unsymmetrical cyanine dye compound of thepresent invention. Typically the solution further contains a bufferingcomponent wherein the buffering component is preferably tris acetate ortris borate. The organic solvent is typically an alcohol or DMSO. Thekit may contain the staining solution as a concentrate or a 1× ready touse concentration.

Kits may further comprise InstaStain papers, such as those provided byEdvotek (Bethesda, Md.) including any article disclosed in EP1057001 andWO9942620, wherein the staining solution has been dried down on orimpregnated into the papers. The paper is then applied to the gelwherein the dye in the InstaStain paper is transferred to the gel. Thekits may further comprise polymerized agarose, either in the form of aprecast gel, such as E-gels (Ethrog/Invitrogen, including any geldisclosed in U.S. Pat. No. 6,562,213; 5,865,974; 5,582,702; 6,379,516;Published U.S. Patent Application 20020134680 and US2002/0112960 andpublished PCT application WO 96/34276 and WO 97/41070), or in a formthat needs to be liquefied and then poured into an appropriate gel slab,such as the Gel-0 Shooters sold by Continental Laboratory Products (SanDiego, Calif.) or the Heat and Pour Agarose sold by IPM Scientific(Eldersburg, Md.). In this instance, the staining solution may bepremixed with the polymerized agarose, added during the liquid phase oradded after polymerization. In another aspect the kit contains a tabletof agarose that needs to have buffer added and then poured into a slab,such as the agarose tablets sold by Bioline (Randolf, Mass.). Thestaining solution may be premixed in the tablet or provided in aseparate vial to be added to the agarose at a step determined by the enduser.

For a kit that is not hazardous to the end user, non-genotoxic, and isused to detect nucleic acid immobilized in a polymeric gel the stainingsolution in the kit typically contains the organic solvent DMSO, thebuffer tris acetate or tris borate and compound 1.

A kit of the invention may optionally further comprise nucleic acidfragments to be used as size markers, controls, additional detectionreagents such as dye compounds specific for only DNA or specific onlyfor RNA.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLES Example 1 Detection of DNA in an Agarose Gel with ThiazoleOrange and Compound 1

Different concentrations of DNA (62.5 ng, 31.25 ng, 15.63 ng, 7.813 ng,3.906 ng, 1.953 ng, 976.6 pg, 488.3 pg, 244.1 pg, 122.1 pg, 61.04 pg and30.52 pg) were loaded and separated on a 1% agarose gel at 80 v in0.5×TBE. The gels were stained with a staining solution comprising TBE(50 mL) and either thiazole orange or compound 1 (4.54 μl of dye stocksolution in DMSO). Alternatively, the gels were prepared to containeither thiazole orange or Compound 1 wherein 1 g of agarose, 100 ml of0.5×TBE was mixed with either 9.08 μl of thiazole orange or Compound 1stock solution.

The DNA was loaded and separated in 0.5×TBE at 80 v. Some of these gelswere also post stained with staining solution. All gels weresubsequently photographed. These gels demonstrate the ability ofthiazole orange and a derivative thereof to detect nucleic acidseparated and immobilized in a gel. See, FIG. 1.

Example 2 Salmonella/Mammalian Microsomal Reverse Mutation Assay (AmesTest)

The Ames assay was performed using the method described by Ames (Ames etal Mutation Research 31 (1975) 347-364; Levin et al. PNAS 79 (1982)7445-7449; Maron and Ames, Mutation Research 113 (1983) 173-215). Testerstains used were Salmonella typhimurium histidine auxotrophs TA97a,TA98, TA100, TA102, TA1535, TA1537 and TA1538. The assay was performedwith test compounds (Ethidium bromide, thiazole orange and Compound 1)at six doses in both the presence and absence of S9 (rat liver extract),along with appropriate vehicle and positive controls (Singer et al.Mutation Research 439 (1999) 37-47). The test compounds, test vehicleand S9 (when appropriate) were combined with molten agar and thenoverlaid over a minimal agar plate. Following incubation at 37° C.,revertant colonies were counted. When S9 was not used, 100 μl of testerstrain and 50 μl of control or test compound were added to 2.5 ml ofselective top agar. When S9 was used, 500 μl of S9 mix, 100 μl of testerstrain and 50 μl of control or test compound was added to 2.0 ml ofselective top agar. The top agar was then overlaid onto the surface of25 ml of minimal bottom agar contained in a 15×100 mm Petri dish. Theinverted plates were incubated for 52±4 hr at 37±2° C.

The number of revertants was counted and the test compounds were eitherconsidered non-mutagenic or mutagenic. The criteria for determining if atest compound was mutagenic was based on a 2-fold increase in meanrevertants per plate for at least one tester strain (TA97a, TA98, TA100and TA102) over the mean revertants per plate of the appropriate vehiclecontrol. For tester strains TA1535, TA1537 and TA 1538a positive mutantwas identified by a 3-fold increase in mean revertants per platecompared to the appropriate vehicle control. In addition, the increasein the mean number of revertants per plate needed to be accompanied by adose response to increasing concentrations of the test compound. Basedon this scoring methodology, in the presence of S9, Compound 1 wasconsidered mild or non-mutagenic for all tester strains wherein Compound1 demonstrated between a 3- and 4-fold increase in revertants for fourof the tester strains, thiazole orange was considered mutagenic in thepresence of S9 with five of the tester strains and ethidium bromide wasconsidered mutagenic for three of the tester strains demonstrating a 4-to 80-fold increase in revertants for four of the tester strains, SeeFIG. 2 and Table 1. Thiazole orange was also considered mutagenic fortwo of the tester strains in the absence of S9. Thus, compared toethidium bromide, thiazole orange is 3-4 times less mutagenic andCompound 1 is 4-5 times less mutagenic. Ethidium bromide and Sybr GreenI had previously been tested wherein Sybr Green I was considered a weakmutagen (Singer et al. Mutation Research 439 (1999) 37-47).

TABLE 2 increase in revertants compared to vehicle control (DMSO) TA97aTA98 TA100 TA102 TA1535 TA1537 TA1538 Ethidium bromide 4.4 68.0 1.6 2.01.4 15 80 Thiazole orange 6.9 6.4 2.2 4.7 1.5 17.4 7.8 Compound 1 3.33.0 1.7 3.7 1.8 1.8 3.7

Example 3 In Vitro Transformation of Syrian Hamster Embryo (SHE) Cellsby 7-Day Exposure Screening Assay

This assay design is based on procedures described by Kerchaert et alMutation Research 356 (1996) 65-84, and is an accepted method forevaluating the carcinogenic potential of chemical substances. Thus, theobjective of the assay was to determine the ability of the testcompounds (Ethidium bromide, thiazole orange and Compound 1) forinducing an increase in morphological transformation of cultured Syrianhamster embryp cells, relative to vehicle control cultures, following a7-day exposure period.

SHE cell cultures were grown in LeBoeuf's modification (0.75 g/L NaHCO3,pH 6.65-6.75) of Dulbecco's Modified Eagle Medium (DMEM) supplementedwith 20% fetal bovine serum (FBS) and 4 mM L-glutamine. The cultureswere maintained at 37±1° C. in an atmosphere of 10±0.5% CO₂ inhumidified air. The known procarcinogen, benzo[a]pyrene (B[a]P) was usedas a positive control, dissolved in DMSO and used at a concentrationrange of 1.25 to 5 μg/ml in the SHE cell cultures. The finalconcentration of DMSO in the cell cultures was about 0.2%. The testcompounds were dissolved in DMSO and used at a final concentration rangeof 0.0400 to 0.800 μg/ml in the SHE cell cultures. After the 7-dayincubation period, the culture dishes were washed in Hanks' balancedsalt solution (HBSS), fixed with methanol, and stained with 10% bufferedaqueous Giemsa. After washing with tap water the dishes were air-dried.The average number of colonies per dish were determined and for eachdose group, the average relative plating efficiency (relative survival,RPE) was calculated, relative to the vehicle control group. The criteriaapplied to identifying colonies showing the morphologically transformedphenotype was 1) colonies possessing piled-up cells with randomorientation (criss-crossing) of the 3-deminesional growth, 2) colonieswith criss-cross cells and increased cytoplasmic basophilia throughoutthe colony, and/or 3) colonies containing cells with decreasedcytoplasm:nucleus ratios compared to normal SHE cells.

The test compounds were evaluated as positive in this assay if theycaused a statistical significant increase in morphologicaltransformation frequency for at least two dose levels compared toconcurrent vehicle control or if one dose showed a statisticallysignificant increase and the trend test was significant. The testcompounds were evaluated as negative if no statistically significantincrease in morphological transformation was obtained. Based on thismethodology, Compound 1 was considered negative while Thiazole orangeand ethidium bromide were both considered positive in the screening SHEcell transformation assay under 7-day exposure conditions.

Specifically, Compound 1 was essentially noncytotoxic at 0.0500 μg/mL(120% RPE), slightly cytoxic at 0.150 μg/mL (88% RPE) and moderatelycytotoxic at 0.300 μg/mL (59% RPE) wherein none of the three treatmentgroups induced a significant increase in the frequency of morphologicaltransformation compared to the concurrent vehicle control.

TABLE 3 Treatment group MT Frequency (%) RPE Vehicle control 0.106 100%(DMSO) Positive Control 1.553 114% Compound 1 0.442 120% 0.0400 μg/mL 0.150 μg/mL 0.315  88%  0.300 μg/mL 0.144  59% MT = morphologicallytransformed; RPE = relative plating efficiency

Thiazole orange was essentially noncytoxic at 0.0400 μg/mL (97% RPE),moderately cytotoxic at 0.150 μg/mL (52% RPE) and highly toxic at 0.260μg/mL (25% RPE) wherein two of the three treatment groups, 0.0400 and0.150 μg/mL, induced significant increases in frequency of morphologicaltransformation compared to concurrent vehicle control.

TABLE 4 Treatment group MT Frequency (%) RPE Vehicle control 0.059 100%(DMSO) Positive Control 1.443  86% Compound 1 0.731  97% 0.0400 μg/mL   0.150 μg/mL 0.852  52%  0.260 μg/mL 0.294  25% MT = morphologicallytransformed; RPE = relative plating efficiency

Ethidium bromide was slightly cytotoxic at 0.200 μg/mL (85% RPE),moderately cytotoxic at 0.400 μg/mL (66% RPE) and highly cytotoxic at0.800 μg/mL (28% RPE). Two of the three treatment groups, 0.400 and0.800 μg/mL, induced significant increases in the frequency ofmorphological transformation compared to the concurrent vehicle control.

TABLE 5 Treatment group MT Frequency (%) RPE Vehicle control 0.059 100%(DMSO) Positive Control 1.443  86% Compound 1 0.313  85% 0.200 μg/mL  0.400 μg/mL 1.304  66% 0.800 μg/mL 0.635  28% MT = morphologicallytransformed; RPE = relative plating efficiency

Example 4 L5178Y TK+/−Mouse Lymphoma Forward Mutation Screen

This assay evaluated the test compounds for their ability inducesignificant mutagenic activity at the thymidine kinase (TK) locus inL5178Y mouse lymphoma cells as assayed by colony growth in the presenceand absence of S9, an exogenous metabolic activation system of mammalianmicrosomal enzymes derived from Acrolor-induced rat liver and is basedon the assay reported by (Clive and Spector, 31 Mutation Research (1975)17-29; Clive et al. 59 Mutation Research (1979) 61-108; Amacher et alMutation Research 72 (1980) 447-474; Clive et al. Mutation Research 189(1987) 143-156). The cell cultures were scored for both cytotoxicity andincreases in the mutant frequency wherein a positive result was based ona frequency that was at least twice the average mutant frequency of theconcurrent vehicle control (DMSO). The mouse lymphoma cells used forthis assay were heterozygous at the TK locus and may undergo a singlestep forward mutation to the TK^(−/−) genotype in which little or no TKactivity remains. These mutants are viable in normal cell culture mediumbut these mutants are resistant to the thymidine analog5-trifluorothymidine (TFT) because they cannot incorporate the toxicanalog of thymidine into DNA. Thus, cells that grow to form colonies inthe presence of TFT are therefore assumed to have mutated, eitherspontaneously or by the test compounds. The results of this assay arenot definitive but rather an indicator that a test compound hasmutagenic properties, or not.

The mouse lymphoma cells were cultured in RPMI 1640 supplemented withhorse serum (10% by volume), Pluronic F68, L-glutamine, sodium pyruvate,penicillin and streptomycin (Amacher et al Mutation Research 72 (1980)447-474; Clive and Spector, Mutation Research 31 (1975) 17-29).Treatment medium was Fisher's medium with the same medium supplements asused for the culture medium except that the horse serum was reduced to5% by volume. Cloning medium was RPMI 1640 with up to 20% horse serum,without Pluronic F68 and with the addition of 0.24% BBL agar to achievea semisolid state. Selection medium was cloning medium containing 3μg/ml of TFT (Clive et al. Mutation Research 189 (1987) 143-156).

The positive controls were Methyl methanesulfonate (MMS) andMethylcholanthrene (MCA) to be used without and with the S9 activation,respectively. MMS is a direct acting mutagen that is highly mutagenic toL5178Y TK^(+/−) cells and was used at a concentration of 13 μg/mL. MCArequires metabolic activation by microsomal enzymes to become mutagenicto L5178Y TK^(+/−) cells, S9, and was used at a concentration of 2and/or 4 μg/mL. The test compounds, ethidium bromide, thiazole orangeand Compound 1, were assayed at concentrations of 0.00625 to 4.93 μg/mL.

The cells were pelleted and resuspended in treatment medium containingcontrols or test compounds, with and without S9. The tubes were placedin an orbital shaker incubator at 35-38° C. and rotated at 70±10orbitals per minute. After a four-hour exposure period the cells werewashed twice, resuspended in 10 mL of culture medium and returned to theorbital shaker and the cells were allowed to grow for two days formutant recovery. Cell densities less than approximately 3×10⁵ cells/mLafter day 2 were no considered for mutant selection. The mutants wererecovered by plating a total of 3×10⁶ cells in selection medium in softagar. The dishes were incubated for 10 to 14 days at approximately 37°C. with about 5% CO₂/95% humidified air.

The mutant frequency was calculated as the ratio of the total number ofmutant colonies found in each mutant selection dishes to the totalnumber of cells seeded, adjusted by the absolute selection cloningefficiency. The cytotoxicity was based on the relative suspension growthof cells over the 2-day expression period multiplied by the relativecloning efficiency at the time of selection resulting in a relativetotal growth (RTG) number. Based on this methodology, all three testcompounds were considered non-mutagenic but with possessing varyingdegrees of cytotoxicity.

Specifically, ethidium bromide, without S9 in the treatment medium, wasweakly cytotoxic at 0.620 μg/mL, moderately cytotoxic at 2.47 μg/mL andmoderately high cytotoxic at 4.93 μg/mL. These concentrationsdemonstrated no increase in the mutant frequency that exceeded theminimum criterion, 2-fold increase compared to the concurrent vehiclecontrol. Ethidium bromide, with S9 in the treatment medium, wasmoderately cytotoxic at 2.47 μg/mL (37.1% RTG) and moderately highcytotoxic at 4.93 μg/mL (23.1% RTG). No increases in the mutationfrequency were observed that exceeded the minimum criterion.

TABLE 6 Without S9 With S9 Mutant Mutant Frequency Frequency TreatmentGroup RTG % (X 10⁻⁶ Units) RTG % (X 10⁻⁶ Units) Vehicle control 99.757.3 98 49.2 (DMSO) Positive Control 22.4 425.6 N/A N/A (MMS 13 μg/mL)Positive Control N/A N/A 35.0 446.4 (MCA 2 μg/mL) Positive Control N/AN/A 17.1 478.4 (MCA 4 μg/mL) Test Compound 53.6 64.3 N/A N/A .620 μg/mL1.24 μg/mL 46.3 59.3 N/A N/A 2.47 μg/mL 33.7 67.6 37.1 73.7 4.93 μg/mL22.7 94.2 23.1 93.6

Compound 1 without S9 was noncytoxic at 0.125 μg/mL (80.3% RTG) andmoderately high cytotoxic at 0.250 μg/mL (26.5% RTG). No increases inthe mutant frequency were observed that exceeded twice the frequency ofthe concurrent vehicle control. With S9, Compound 1 was weakly cytotoxicat 1.24 μg/mL (65.1% RTG), moderately cytotoxic at 2.47 μg/mL (47.1%RTG) and was excessively cytotoxic at 4.93 μg/mL (7.6% RTG). Noincreases in the mutation frequencies were observed that were twice thefrequency of the concurrent vehicle control.

TABLE 7 Without S9 With S9 Mutant Mutant Frequency Frequency RTG % (X10⁻⁶ Units) RTG % (X 10⁻⁶ Units) Vehicle control 100.5 49.0 98 72.6Positive Control 27 311.6 N/A N/A (MMS 13 μg/mL) Positive Control N/AN/A 35.0 446.4 (MCA 2 μg/mL) Positive Control N/A N/A 17.1 478.4 (MCA 4μg/mL) Test Compound 80.3 55.2 N/A N/A 0.125 μg/mL 0.250 μg/mL 26.5 67.3N/A N/A  1.24 μg/mL N/A N/A 65.1 70.6  2.47 μg/mL N/A N/A 47.1 86.7 4.93 μg/mL N/A N/A 7.6 114.5

Thiazole orange, without S9, was weakly cytotoxic ar 0.100 μg/mL (60.9%RTG) and moderately cytotoxic at 0.200 μg/mL (23.4% RTG). No increasesin mutation frequency were observed that were twice the frequency of theconcurrent vehicle control. With S9, thiazole orange was noncytoxic at4.93 μg/mL (90.4% RTG) and moderately high cytotoxic at 9.85 μg/mL(22.6% RTG). No increases in the mutant frequency were observed thatwere twice the frequency of the concurrent vehicle control.

TABLE 8 Without S9 With S9 Mutant Mutant Frequency Frequency TreatmentGroup RTG % (X 10⁻⁶ Units) RTG % (X 10⁻⁶ Units) Vehicle control 100.549.0 98 72.6 Positive Control 27 311.6 N/A N/A (MMS 13 μg/mL) PositiveControl N/A N/A 35.0 446.4 (MCA 2 μg/mL) Positive Control N/A N/A 17.1478.4 (MCA 4 μg/mL) Test Compound 60.9 53.0 N/A N/A 0.100 μg/mL 0.200μg/mL 23.4 58.8 N/A N/A  4.93 μg/mL N/A N/A 90.4 77.4  9.85 μg/mL N/AN/A 22.6 78.0

Example 5 Screening Assay for Chromosomal Aberrations in Cultured HumanPeripheral Blood Mononuclear Cells (PBMC)

The objective of this assay was to evaluate the ability of the testcompounds, ethidium bromide, thiazole orange and Compound 1, to causestructural chromosomal aberrations in cultured human lymphocytes withand without exogenous metabolic activation system. Human venous bloodfrom healthy adult volunteers was drawn in heparinized vacutainers. Thewhole blood cultures were initiated in 15 ml centrifuge tubes by addingapproximately 0.3 ml of fresh heparinized blood into a sufficient volumeof culture medium, to that the final volume was 5 mL in the assay withand without metabolic activation after the addition of the testcompound. The culture medium was RPMI 1640 supplemented withapproximately 20% heat-inactivated fetal bovine serum (FBS), penicillin(100 units/mL), streptomycin (100 μg/mL), L-glutamine (2 mM) and 2%phytohemagglutinin M (PHA-M). The cultures were incubated with loosecaps at 37° C.±2° C. in a humidified chamber of approximately 5% CO2 inair.

The positive controls were Mitomycin C (MMC) and cyclophosphamide (CP)to be used without and with the S9 activation, respectively. MMC is adirect acting clastogen that does not require metabolic activation andwas used at a concentration of 0.025 to 3.0 μg/mL. CP requires metabolicactivation by microsomal enzymes to become converted to a clastogenicintermediate, and was used at a concentration of 10 to 300 μg/mL. Thetest compounds, ethidium bromide, thiazole orange and Compound 1, wereassayed at concentrations of 0.500 to 10 μg/mL. The in vitro metabolicactivation system consisted of a rat liver post-mitochondrial fraction(S9) and an energy-producing system (NADPH plus isocitric acid) (Maronand Ames, 113 Mutation Research (1983) 173-215).

Two days after culture initiation, the cultures were treated with thetest compounds. The cultures without the S9 metabolic activation mixturewere incubated for an additional 22 hours with Colcemid (0.1 μg/mL)added for the last 2±0.5 hours. The cultures with the S9 metabolicactivation mixture were incubated for a 3-hour exposure period. Afterexposure the cells were washed at least twice with PBS, and freshculture medium added. The cell culture was then incubated for anadditional 18 hours, with Colcemid (0.1 μg/mL) added for the last 2±0.5hours of incubation.

At the end of the incubation period the cultures were centrifuged, thesupernatant discarded, and the cells swollen with 75 mM KCl, fixed inmethanol:glacial acetic acid (3:1 v/v), dropped onto glass slides andair dried. The slides were stained with 5% Giemsa and air dried and thenanalyzed for mitotox index, chromosomal aberrations including polyploidyand endoreduplication. Based on this methodology, the cells individuallytreated with the three test compounds, with and without the S9activation mixture, showed no significant increase in the number ofcells with structural aberrations, polyploidy or endoreduplicationcompared to the concurrent vehicle control. Thus, ethidium bromide,thiazole orange and Compound 1 were considered negative for inducingstructural chromosomal aberrations with and without metabolicactivation.

Example 6 Comparison of Ethidium and Compound 1 Staining of Nucleic AcidSeparated and Immobilized in an Agarose Gel

Different concentrations of DNA (62.5 ng, 31.25 ng, 15.63 ng, 7.813 ng,3.906 ng, 1.953 ng, 976.6 pg, 488.3 pg, 244.1 pg, 122.1 pg, 61.04 pg and30.52 pg) were loaded and separated on a 1% agarose gel at 60 v in0.5×TBE. The gels were stained with a staining solution comprising TBE(50 mL) and either ethidium bromide (2.5 μl of stock solution, 10 mg/mLin water) or compound 1 (5 μl of dye stock solution in DMSO for a finalconcentration of 1 μM) for 30, 60 and 90 minutes. All gels weresubsequently photographed. These gels demonstrate that Compound 1 is atleast as sensitive as ethidium bromide for detection nucleic acid in agel using similar staining procedures.

Example 7 Hazardous Waste Screening Test

Compound 1 was tested to determine whether or not the compound washazardous or toxic to aquatic life. Ten fathead minnows (Pimephakespromelas) were placed each in a 8 liter tank containing vehicle controland a concentration of Compound 1 at 250 mg/L, 500 mg/L and 750 mg/L.After a 96 hour exposure period the number of viable minnows werecounted. The survival rate of the minnows for the control and Compound 1was 100%. Thus, compound 1 has a LC₅₀ value >500 mg/L, which isclassified as not hazardous under CCR Title 22 acute toxicity to aquaticlife.

Example 8 Cell Permeability of Compound 1 on Live Eukaryotic Cells

MRC5 human lung fibroblast cells were harvested and grown in completeculture media (DMEM+10% FBS) for one day after seeding coverslips. Cellswere then removed from complete media and placed in Hank's balanced saltsolution w/sodium bicarbonate (HBSS) supplemented with 5 mM HEPES, 100uM L-glutamine and 100 uM succinate containing varying concentrations ofCompound 1. Concentrations tested included 0.5, 1.0, 5.0 and 10.0 μM.Cells were incubated for 5 minutes at 37° C./5% CO2. Cells were washed3×30 seconds in HBSS and mounted on microscope slides in HBSS and sealedwith paraffin. After mounting, slides were examined on a Nikon Eclipse800 upright fluorescent microscope and imaged with standard FITC andTRITC filter sets, a Princeton Instrument MicroMax cooled CCD camera,and Universal Imaging MetaMorph imaging software. Compound 1 appears tobe cell permeable in live MRC5 cells. Labeling pattern appears to benuclear and cytoplasmic with prominent signal in the nucleolus oflabeled cells. Signal associated with Compound 1 is detectable with bothFITC and TRITC filter sets with greater signal intensity using the FITCset. Regardless of concentration, nucleolar labeling seems to be themost prominent and appears to become slightly more prominent at lowerconcentrations. Off cell background was minimal in all cases.

Example 9 Detection of DNA in an Agarose Gel with Compound 1

Different quantities of the Low DNA Mass Ladder 1 μl, 0.5 μl, 0.25 μl,0.13 μl, (Invitrogen Corp. Cat #10068-013) were loaded on an E-Gel 2%(Invitrogen), prepared according to the description in U.S. Pat. No.5,582,702 where the Ethidium Bromide has been replaced by a 4×concentration of Compound 1 from a 10000× solution. The gels were runusing the Powerbase (Invitrogen, Cat #G6200-04) for 30 minutes, thenvisualized using the Clare Chemical Dark Reader. See, FIG. 4.

Example 10 EPA Acute Oral Toxicity Test for Compound 1 in 0.5×TBE

A Limit Screen test was performed according to OPPTS guidelines(870.1100) using three female Sprague Dawley rats, which received anoral Limit Dose of 5000 mg/kg of the test article. The animals wereobserved for mortality, weight change and toxic signs for a two weekperiod.

Since all three rats survived for two weeks after the doseadministration, the LD₅₀ for the test article was considered to begreater than the limit dose and no additional testing was required.

All animals were euthanized at the termination of the study. Grossnecropsies were performed and no abnormalities were observed in any ofthe test animals.

Example 11 NPDES (National Pollutant Discharge Elimination System)Testing for Compliance with the Clean Water Act

Compound 1 complies with the Clean Water Act and the National PollutantDischarge Elimination System regulations, as it does not containcyanide, phenolics, pollutant metals, organochlorine pesticides, PCBs,or semi-volatile or volatile organic compounds. The testes wereperformed according to EPA protocols cited in Table 9.

TABLE 9 Analysis Compound 1 in (EPA method, as per 40 CFR part 136) 0.5XTBE 0.5 XTBE pH (150.1) 8.45 8.48 Total Cyanide (335.2) None None BOD(405.1) None None COD (410.1) 7020 6840 Ammonia as Nitrogen (350.1) 253248 Total Organic Carbon (415.1) 2480 2360 Total Phenolics (420.1) NoneNone Organochlorine Pesticides and PCBs None None (608M) Semi-volatileOrganic Compounds (625) None None Volatile Organic Compounds (624) NoneChloroform (17 ug/L) Metals (Sb, As, Be, Cd, Cr, Cu, Pb, Hg, None NoneNi, Se, Ag, Tl, Zn) (6010B, 7060A, 7421, 7470A, 7740, 7841)

Example 12 Synthesis of Compound 1

A mixture of 33.92 g of lepidine and 50.45 g of propyl tosylate isheated at 110 C for 1 hour. The reaction is cooled to room temperatureand 600 mL of ethyl acetate is added and heated at 60 C for 1 hour. Themixture is filtered and 75.75 g of the intermediate4-methyl-1-propylquinolinium tosylate is obtained. The intermediate ismixed with 78.05 g of 3-methyl-2-methylthiobenzothiazolium tosylate in300 mL of methylene chloride and 64.53 g of triethylamine is introduced.The reaction mixture is stirred at room temperature overnight and 1 L ofethyl acetate is then added and the product is filtered.

The preceding examples can be repeated with similar success bysubstituting the specifically described nucleic acid dye compounds ofthe preceding examples with those generically and specifically describedin the forgoing description. One skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt to various usages andconditions.

All patents and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference.

What is claimed is:
 1. A compound for the detection of nucleic acidimmobilized on a solid or semi solid support, wherein the compound is anunsymmetrical cyanine dye compound that is characterized as beingessentially non-genotoxic in eukaryotic cells.
 2. The compound accordingto claim 1, wherein the compound is in an aqueous solution.
 3. Thecompound according to claim 1, wherein the compound is immobilized on orin a solid or semi-solid matrix.
 4. The compound according to claim 3,wherein the solid or semi-solid matrix comprises agarose.
 5. Thecompound according to 3, wherein the solid or semi-solid matrix is apolymeric membrane.
 6. The compound according to claim 1, wherein thecyanine compound has the formula


7. A kit for the detection of immobilized nucleic acid, the kitcomprising: a. a compound having the formula

wherein X is O, S or C(CH₃)₂; R¹ is a fused benzene, methoxy, a C₁-C₆alkyl; R² and R³ are independently a C₁-C₆ alkyl; R⁴ is a C₁-C₆ alkyl ora methoxy; t is independently 0, 1, 2, 3, or 4; s is independently 0, 1,2, 3 or 4 and n is 0, 1, 2 or 3; with the proviso that the cyanine dyecompound is not thiazole orange.
 8. The kit according to claim 7,wherein the compound is in an aqueous solution.
 9. The kit according toclaim 8, wherein the aqueous solution comprises tris borate or trisacetate.
 10. The kit according to claim 7, wherein the compound isdissolved in an alcohol or DMSO.
 11. The kit according to claim 7,wherein the compound is immobilized on or in a solid or semi-solidmatrix.
 12. The kit according to claim 11, wherein the matrix comprisesagarose.
 13. The lit according to claim 11, wherein the matrix is apolymeric membrane.
 14. The kit according to claim 7, wherein thecompound is

and is characterized as being essentially non-genotoxic.